HA Gene Research Articles

Multiple introductions of reassorted highly pathogenic avian influenza viruses (H5N8) clade 2.3.4.4b causing outbreaks in wild birds and poultry in Egypt

Recently, an increased incidence of outbreaks of highly pathogenic avian influenza (HPAI) H5N8 in poultry linked to infected migratory birds has been reported from different European, Asian and African countries.In Egypt, incursion of HPAI H5N8 virus of clade 2.3.4.4b has been recently registered. Full genomic characterization of 3 virus isolates from wild birds and poultry (backyard and commercial farm sectors) showed high nucleotide similarity among the HA, NA, M, and NS gene segments of the three Egyptian HPAI H5N8 viruses, indicating that they are descendants of a common ancestral virus. However, the analyzed Egyptian H5N8 viruses revealed distinct genotypes involving different origins of the PB2, PB1, PA and/or NP segments. In genotype-1 represented by strain A/common-coot/Egypt/CA285/2016 the PB2 and NP segments showed closest relationship to H5N6 and H6N2 viruses, recently detected in Italy. The second is replacement of PB1 and NP genes A novel reassortant, represented by strain A/duck/Egypt/SS19/2017, showed an exchange of PB1 and NP genes which might have originated from H6N8 or H1N1 and H6N2 viruses. Finally, replacement of PA and NP genes characterized strain A/duck/Egypt/F446/2017. Bayesian phylogeographic analyses revealed that Egyptian H5N8 viruses are highly likely derived from Russian 2016 HPAI H5N8 virus (A/great_crested_grebe/Uvs-Nuur_Lake/341/2016 (H5N8)) and the reassortment likely occurred before incursion to Egypt.

Vaccine-mismatched influenza B/Yamagata lineage viruses in Cuba, 2012–2013 season

Annual trivalent influenza vaccines contain one of influenza B lineages; influenza B/Victoria-lineage or influenza B/Yamagata viruses. Theoretically, these vaccines should protect against viruses expected to circulate in the next influenza season. The National Influenza Centers, based on surveillance data from National Reference Laboratories, selects the strains composing each annual trivalent or tetravalent vaccine.Nevertheless, in some epidemics, vaccine strains do not match genetically with circulating strains. The aim of the present study is to compare the HA1-domain of 42 influenza B viruses circulating in Cuba during the 2012–2013 season with the vaccine strain B/Wisconsin/01/2010-like virus from the B/Yamagata lineage, included in the 2012–2013 Northern-Hemisphere Influenza vaccine. The efficacy of the influenza vaccine was also estimated. The analysis of the present study indicates that the B/Victoria and B/Yamagata lineages co-circulated in Cuba in the 2012–2013 season. In 2012–2013 season, according to the sequences analysis, trivalent vaccine did not match with the circulating strains. The present study also detected amino acid substitutions which could have altered the antigenic properties of HA gene. The results presented here suggest the need to consider a possible introduction of tetravalent influenza vaccine in Cuba, as has been recommended by the WHO to ensure higher levels of protection.

Highly Pathogenic Avian Influenza H5N1 in Chickens in Upper Egypt

One-year surveillance of HPAI (H5N1) virus infection in different chicken flocks (141 flock) from different Governorates in Upper Egypt was carried out, during the period from January 2015 to January 2016. The detection rate of highly pathogenic avian influenza (HPAI) H5N1 virus was 21.3% (30/141) which was highest during the winter season compared with the other seasons. The investigated flocks suffered from respiratory and nervous signs and cyanosis of the comb and wattles was observed in some birds as well as subcutaneous hemorrhages in shanks. The thirty positive H5N1 samples were tested for the existence of other respiratory viruses. The results were as follows: single H5N1 virus infection was only detected in 5 flocks (16.6%). The mixed H5N1 with H9N2 in 2 flocks (6.6%); H5N1with NDV in 12 farms (40%); H5N1 with IBV in 7 farms (23.3%) while H5N1, H9N2 and NDV were detected together in 4 flocks (13.3 %). The sequence analysis of the HA gene from five selected H5N1 isolates revealed that these strains were clustered with Egyptian classical H5N1 viruses (sub-clade 2.2.1.2) of the Eurasian origin. It is concluded that, the five H5N1 subtype isolates of HPAI are clustered with classical H5N1 viruses to subclade 2.2.1.2 of Eurasian origin. Therefore, vaccination of the backyard chicken as well as chicken farms with suitable homologues vaccine to eliminate HPAI virus from Egypt is recommended.

Emergence of H3N8 equine influenza virus in donkeys in China in 2017

Equine influenza virus is a major respiratory pathogen in horses. Although both horses and donkeys belong to the genus Equus, donkey infection with influenza viruses is rare. In March 2017, an influenza outbreak occurred in donkeys in Shandong province, China. The causative virus, A/donkey/Shandong/1/2017(H3N8), was isolated from a dead donkey. Genetic analysis indicated that the virus originated from influenza A (H3N8) clade 2 of the Florida sub-lineage that has been circulating in Asian equine populations. Comparison of the deduced amino acid sequence of the HA gene of this causative virus with that of the A/equine/Richmond/1/2007 vaccine strain showed that substitutions had occurred in the antigenic regions A, B, and C. This study provides insight into the currently circulating and newly emerging H3N8 strains in donkeys in China.

Crosstalk between H9N2 avian influenza virus and crypt-derived intestinal organoids

The spread of Avian influenza virus via animal feces makes the virus difficult to prevent, which causes great threat to human health. Therefore, it is imperative to understand the survival and invasion mechanism of H9N2 virus in the intestinal mucosa. In this study, we used mouse threedimensional intestinal organoids that contained intestinal crypts and villi differentiated from intestinal stem cells to explore interactions between H9N2 avian influenza virus and the intestinal mucosa. The HA, NA, NP and PB1 genes of H9N2 viruses could be detected in intestinal organoids at 1 h, and reached peak levels at 48 h post-infection. Moreover, the HA and NP proteins of H9N2 virus could also be detected in organoids via immunofluorescence. Virus invasion caused damage to intestinal organoids with reduced mRNA transcript expression of Wnt3, Dll1 and Dll4. The abnormal growth of intestinal organoids may be attributed to the loss of Paneth cells, as indicated by the low mRNA transcript levels of lyz1 and defcr1. This present study demonstrates that H9N2 virus could invade intestinal organoids and then cause damage, as well as affect intestinal stem cell proliferation and differentiation, promoting the loss of Paneth cells.

The M2 protein of live, attenuated influenza vaccine encodes a mutation that reduces replication in human nasal epithelial cells.

The influenza A virus components of the live, attenuated influenza vaccine (LAIV) encode the HA and NA gene segments from a circulating virus strain and the remaining gene segments from the cold-adapted master donor virus, A/Ann Arbor/6/1960 (H2N2). The master donor virus imparts at least three phenotypes: temperature-sensitivity (ts), attenuation (att), and cold-adaption (ca). The genetic loci responsible for the att and ts phenotypes of LAIV were mapped to PB1, PB2, and NP by reverse genetics experiments using immortalized cell lines. However, some in vivo studies have demonstrated that the M segment, which acquired an alanine (Ala) to serine (Ser) mutation at M2 position 86 during cold adaption - a mutation found in no other influenza A virus strain - contributes to the att phenotype. Prior studies have shown this region of the M2 cytoplasmic tail to be critical for influenza virus replication. Using reverse genetics, we demonstrate that certain amino acid substitutions at M2 positions 83 and 86 alter the replication of influenza A/Udorn/307/72 (H3N2). Importantly, substitution of a Ser at M2 position 86 reduces A/Udorn/307/72 replication in differentiated primary human nasal epithelial cell (hNECs) cultures, but does not considerably affect replication in MDCK cells. When a Ser was substituted for Ala at M2 86 in LAIV, the virus replicated to higher titers and with faster kinetics in hNEC cultures, implicating this amino acid change as contributing to LAIV attenuation. Increased replication also resulted in increased production of IFN-λ. These data indicate the LAIV associated Ser mutation at M2 position 86 contributes to the att phenotype and is associated with a differential regulation of interferon in LAIV infection.

Molecular epidemiology of swine influenza A viruses in the Southeastern United States, highlights regional differences in circulating strains

Swine influenza A virus (IAV) can cause widespread respiratory disease with high morbidity, low mortality, and have a substantial economic impact to the swine industry. Swine infection may contribute to pandemic IAV given their susceptibility to both avian and human IAVs. Currently, three IAV subtypes (H1N1, H3N2 and H1N2) circulate in swine in North America frequently combining gene segments from avian or human viruses. This study investigated the prevalence of IAV in commercial swine herds. A total of 1878 oral fluid samples were collected from pigs of all ages from 201 commercial farms located in North Carolina and South Carolina. Sixty-eight oral fluid samples from 35 farms were positive by MP gene PCR with an overall IAV-positivity of 3.6%. On the herd level, the percentage of IAV positivity was 17.4%. Fifty-six viruses were subtyped, while 12 were partly subtyped or not subtyped at all. Using de novo assembly, complete sequences were obtained for 59 HA genes. The majority of IAVs subtyped had an H1 HA demonstrating a considerable prevalence over H3 viruses. Furthermore, only six out of eleven HA types were detected which has implications for the selection of vaccines used by swine producers in the region.

Cloning and prokaryotic expression of the globular head domain of hemagglutinin antigen (HA1) of influenza A (H3N2) virus in Escherichia coli and Bacillus subtilis

Background: The influenza virus hemagglutinin is the major surface protein of the influenza A virus which is composed of HA1 and HA2 subunits. HA1 has an important role in binding of virus to cells and designing neutralizing antibodies. Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) both are known as the most useful prokaryotic hosts to express recombinant proteins. The aim of this study was to clone and express recombinant HA1protein in E. coli and B. subtilis bacteria.Materials and Methods: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli. The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Results: HA1 gene was cloned into pET-28a vector and pHT43 shuttle vector and then, both transformed to E. coli.The recombinant plasmids were extracted and then transformed into the BL21 and WB600 as expressing hosts. After induction with isopropyl-β-d-thiogalactoside (IPTG), expressed recombinant protein was analyzed by SDS-PAGE. Finally, the expressed protein was confirmed by the Western blot.Conclusion: This study demonstrated a strategy for production and purification of recombinant protein in large scale to test as vaccine candidate against influenza and it’s potentially immunogenicity be assessed in animal models.

Internal Gene Cassette from a Genotype S H9N2 Avian Influenza Virus Attenuates the Pathogenicity of H5 Viruses in Chickens and Mice.

H9N2 avian influenza virus (AIV) of genotype S frequently donate internal genes to facilitate the generation of novel reassortants such as H7N9, H10N8, H5N2 and H5N6 AIVs, posing an enormous threat to both human health and poultry industry. However, the pathogenicity and transmission of reassortant H5 viruses with internal gene cassette of genotype S H9N2-origin in chickens and mice remain unknown. In this study, four H5 reassortants carrying the HA and NA genes from different clades of H5 viruses and the remaining internal genes from an H9N2 virus of the predominant genotype S were generated by reverse genetics. We found that all four H5 reassortant viruses showed attenuated virulence in both chickens and mice, thus leading to increased the mean death times compared to the corresponding parental viruses. Consistently, the polymerase activity and replication ability in mammalian and avian cells, and the cytokine responses in the lungs of chickens and mice were also decreased when compared to their respective parental viruses. Moreover, these reassortants transmitted from birds to birds by direct contact but not by an airborne route. Our data indicate that the internal genes as a whole cassette from genotype S H9N2 viruses play important roles in reducing the pathogenicity of the H5 recombinants in chickens and mice, and might contribute to the circulation in avian or mammalian hosts.

Cloning recombinant pHW2000 vector carrying the HA gene for preparation of the primary influenza A/H5N1 vaccine strain

Influenza A/H5N1 virus evolves rapidly and generate new variants, therefore it is essential to develop effective vaccines against the currently circulating influenza strains. Among clades and subclades of highly pathogenic avian influenza (HPAI) H5N1 viruses circulating in Vietnam, H5N1 clade 1.1 and clade 2.3.2.1c possess genetic relationships to many strains of influenza; thus they are suggested to be used for producing vaccines against avian influenza. In this article, two HA gene segments of two types of A/H5N1 influenza clade have been designed: HA clade 1.1 gene consists 1825 nucleotides encoding 565 amino acids, HA clade 2.3.2.1c gene consists 1822 nucleotides, encoding 564 amino acids. Most importantly, nucleotide sequence of the pathogenic region of HA was removed. Each of the two HA segments corresponding to the two clades were successfully cloned into pHW2000 vector and will be used as a candidate for production of avian influenza vaccines using reverse genetics technique.

First genome report and analysis of chicken H7N9 influenza viruses with poly-basic amino acids insertion in the hemagglutinin cleavage site

We report the full-length sequence of two chicken source influenza A (H7N9) viruses found in Guangdong live poultry market (LPM) during the most recent wave of human infections (from October 2016 to the present time). These viruses carry insertion of poly-basic amino acids (KGKRTAR/G) at the protease cleavage site of the HA protein, which were previously found in the highly pathogenic (HP) human influenza A (H7N9) [IAV(H7N9)] strains. Phylogenetic analysis of these two novel avian influenza viruses (AIVs) suggested that their genomes reassorted between the Yangtze River Delta (YRD) and Pearl River Delta (PRD) clades. Molecular clock analysis indicated that they emerged several months before the HP human strains. Collectively, our results suggest that IAV(H7N9) viruses evolve in chickens through antigenic drift to include a signature HP sequence in the HA gene, which highlights challenges in risk assessment and public health management of IAV(H7N9) infections at the human-animal interface.

Vertical Transmission of H9N2 Avian Influenza Virus in Goose.

During a study on high mortality cases of goose embryo in Shandong Province, China (2014–2015), we isolated an H9N2 avian influenza virus (AIV) strain (A/goose/Shandong/DP01/2014, DP01), which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups (A, B, C, D, and E) for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible.

Pathogenicity of the Egyptian A/H5N1 avian influenza viruses in chickens.

Long-term circulation of highly pathogenic avian influenza H5N1 viruses of clade 2.2.1 in Egyptian poultry since February 2006 resulted in the evolution of two distinct clades: 2.2.1.1 represents antigenic-drift variants isolated from vaccinated poultry and 2.2.1.2 that caused the newest upsurge in birds and humans in 2014/2015. In the present study, nine isolates were collected from chickens, ducks and turkeys representing the commercial and backyard sectors during the period 2009-2015. The subtyping was confirmed by hemagglutination inhibition (HI) test, RT-qPCR and sequence analysis. The Mean Death Time (MDT) and Intravenous Pathogenicity Index (IVPI) for all isolates were determined. Sequence analysis of the HA gene sequences of these viruses revealed that two viruses belonged to clade 2.2.1.1 and the rest were clade 2.2.1.2. Antigenic characterisation of the viruses supported the results of the phylogenetic analysis. The MDT of the isolates ranged from 18 to 72h and the IVPI values ranged from 2.3 to 2.9; viruses of the 2.2.1.1 clade were less virulent than those of the 2.2.1.2 clade. In addition, clade-specific polymorphism in the HA cleavage site was observed. These findings indicate the high and variable pathogenicity of H5N1 viruses of different clades and host-origin in Egypt. The upsurge of outbreaks in poultry in 2014/2015 was probably not due to a shift in virulence from earlier viruses.

Phylogeny, Pathogenicity, and Transmission of H5N1 Avian Influenza Viruses in Chickens

We analyzed five H5N1 avian influenza viruses (AIVs) isolated from different birds in 2012 in China. Based on whole-genome sequences, we divided the viruses into four genotypes. The DKE26, GSE43, and DKE53 viruses belonged to Genotypes 1–3, respectively. The CKE93 and CKE96 viruses were classified into Genotype 4. Genotypes 1–3 correspond to the viruses containing the HA gene of clade 2.3.2, and Genotype 4 is the virus that bears the HA gene of clade 7.2. To better understand the pathogenicity and transmission of the viruses, we infected chickens with 103 EID50/0.1 ml GSE43 (clade 2.3.2) or CKE93 (clade 7.2) virus. Our results revealed that 6 of 7 specific-pathogen-free (SPF) chickens inoculated with GSE43 virus were dead before 7-day post-infection, but all the SPF chickens inoculated with CKE93 virus survived the infection. Both the GSE43 and CKE93 viruses replicated systemically in chickens. The virus titers of GSE43 virus in tested organs were obviously higher than those of CKE93 virus. Our results revealed that the pathogenicity and replication of GSE43 in chickens was much higher than those of CKE93. The GSE43 virus could transmit between chickens, but the CKE93 could not transmit between chickens by naïve contact. Therefore, different clades of H5N1 AIVs possessed variable pathogenicities and transmission abilities among chickens. Our study contributes to knowledge of pathogenic variations of prevalent H5N1 viruses.

Phylogenetic analysis of H9N2 avian influenza viruses in Afghanistan (2016-2017).

Avian influenza A virus (AIV) subtype H9N2 is the most prevalent subtype found in terrestrial poultry throughout Eurasia and has been isolated from poultry outbreaks worldwide. Tracheal tissue specimens from 100 commercial broiler flocks in Afghanistan were collected between 2016 and 2017. After real-time RT-PCR, AI-positive samples were further characterized. A part of the HA gene was amplified using RT-PCR and sequenced. The results of real-time RT-PCR showed that 40 percent of the flocks were AI positive. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian-lineage G1 AIVs and had a correlation with H9N2 AIV circulating in the poultry population of the neighboring countries over the past decade. Analysis of the amino acid sequence of HA revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity and specificity for the avian-like SAα2,3 receptor, demonstrating their specificity for and adaptation to domestic poultry. The results of the current study provide great insights into H9N2 viruses circulating in Afghanistan's poultry industry and demonstrate the necessity of planning an applied policy aimed at controlling and managing H9N2 infection in Afghan poultry.

Serological and virological surveillance of avian influenza virus in domestic ducks of the north-east region of Bangladesh

BackgroundWild waterfowl are considered as the natural reservoir for avian influenza (AI) viruses. Bangladesh has been experiencing highly pathogenic avian influenza (HPAI) outbreaks since 2007, mostly in chickens and occasionally in ducks. Ducks play an important role in the persistence and genetic recombination of AI viruses. This paper presents the results of serological and virological monitoring of AI in domestic ducks in 2013 in the north-east region of Bangladesh.ResultsA total of 871 and 662 serum samples and 909 and 302 pairs of cloacal and oropharyngeal swabs from domestic ducks of Mymensingh and Sylhet division, respectively, were analysed. Antibodies to type A influenza virus were detected by blocking ELISA in 60.73 and 47.73% serum samples of Mymensingh and Sylhet division, respectively. On haemagglutination-inhibition (HI) test 17.5% of ELISA positive serum samples were found to be seropositive to H5 avian influenza virus. Five cloacal swabs and one oropharyngeal swab were positive for M gene of type A influenza virus by real time RT-PCR (rRT-PCR), but all of them were negative for H5 influenza virus. Three of the six viruses were successfully characterized as H1N5, H2N5 and H7N5 subtype of AI virus, the other three remained uncharacterized. On sequencing and phylogenetic analysis the HA and NA genes were found to be of Eurasian avian lineage. The H7 virus had cleavage site motif of low pathogenic virus.ConclusionsLow pathogenic avian influenza viruses were detected from apparently healthy domestic ducks. A small proportion of domestic ducks were found seropositive to H5 AI virus.

Live-attenuated auxotrophic mutant of Salmonella Typhimurium expressing immunogenic HA1 protein enhances immunity and protective efficacy against H1N1 influenza virus infection.

To evaluate the efficacy of attenuated Salmonella Typhimurium (JOL912) as a live bacterial vaccine vector. The JOL912 engineered to deliver HA1 protein from influenza A/Puerto Rico/8/1934 (H1N1; PR8) virus was coined as JOL1635 and further evaluated for immunogenicity and protective efficacy. The JOL1635 stably harbored the HA1 gene within pMMP65 plasmid with periplasmic expression and effective delivery of HA1 protein to RAW264.7 cells. The JOL1635 immunized chickens showed the significant increase in HA1-specific IgG, sIgA antibody, IFN-γ, IL-6 cytokine and cellular immune responses. The postoral challenge, the JOL1635-immunized chickens showed a faster clearance of PR8 virus cloacal shedding than the control group. Generated JOL1635 can establish specific immunogenicity and protection against the PR8 virus in chickens.

Frequency of respiratory virus infections and next-generation analysis of influenza A/H1N1pdm09 dynamics in the lower respiratory tract of patients admitted to the ICU.

Recent molecular diagnostic methods have significantly improved the diagnosis of viral pneumonia in intensive care units (ICUs). It has been observed that 222G/N changes in the HA gene of H1N1pdm09 are associated with increased lower respiratory tract (LRT) replication and worse clinical outcome. In the present study, the frequency of respiratory viruses was assessed in respiratory samples from 88 patients admitted to 16 ICUs during the 2014–2015 winter-spring season in Lombardy. Sixty-nine out of 88 (78.4%) patients were positive for a respiratory viral infection at admission. Of these, 57/69 (82.6%) were positive for influenza A (41 A/H1N1pdm09 and 15 A/H3N2), 8/69 (11.6%) for HRV, 2/69 (2.9%) for RSV and 2/69 (2.9%) for influenza B. Phylogenetic analysis of influenza A/H1N1pdm09 strains from 28/41 ICU-patients and 21 patients with mild respiratory syndrome not requiring hospitalization, showed the clear predominance of subgroup 6B strains. The median influenza A load in LRT samples of ICU patients was higher than that observed in the upper respiratory tract (URT) (p<0.05). Overall, a greater number of H1N1pdm09 virus variants were observed using next generation sequencing on partial HA sequences (codons 180–286) in clinical samples from the LRT as compared to URT. In addition, 222G/N/A mutations were observed in 30% of LRT samples from ICU patients. Finally, intra-host evolution analysis showed the presence of different dynamics of viral population in LRT of patients hospitalized in ICU with a severe influenza infection.

Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses.

Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

Molecular Evolution and Emergence of H5N6 Avian Influenza Virus in Central China.

H5N6 avian influenza virus (AIV) has posed a potential threat to public health since its emergence in China in 2013. To understand the evolution and emergence of H5N6 AIV in the avian population, we performed molecular surveillance of live poultry markets (LPMs) in Wugang Prefecture, Hunan Province, in central China, during 2014 and 2015. Wugang Prefecture is located on the Eastern Asian-Australian migratory bird flyway, and a human death due to an H5N6 virus was reported in the prefecture on 21 November 2016. In total, we sampled and sequenced the complete genomes of 175 H5N6 AIVs. Notably, our analysis revealed that H5N6 AIVs contain at least six genotypes arising from segment reassortment, including a rare variant that possesses an HA gene derived from H5N1 clade 2.3.2 and a novel NP gene that has its origins with H7N3 viruses. In addition, phylogenetic analysis revealed that genetically similar H5N6 AIVs tend to cluster according to their geographic regions of origin. These results help to reveal the evolutionary behavior of influenza viruses prior to their emergence in humans.IMPORTANCE The newly emerged H5N6 influenza A virus has caused more than 10 human deaths in China since 2013. In November 2016, a human death due to an H5N6 virus, in Wugang Prefecture, Hunan Province, was confirmed by the WHO. To better understand the evolution and emergence of H5N6 viruses, we surveyed live poultry markets (LPMs) in Wugang Prefecture before the reported human death, with a focus on revealing the diversity and genomic origins of H5N6 in birds during 2014 and 2015. In general, H5N6 viruses in this region were most closely related to H5N1 clade 2.3.4.4, with the exception of one virus with an HA gene derived from clade 2.3.2 such that it represents a novel reassortant. Clearly, the ongoing surveillance of LPMs is central to monitoring the emergence of pathogenic influenza viruses.